JP3473985B2 - Method for producing D-proline - Google Patents

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for producing D-proline. D-proline is a substance useful as a synthetic raw material for medicines, agricultural chemicals and the like.

2. Description of the Related Art As a method for producing D-proline, L-
Ornithine Proteus to Mitajiri (Proteus mitajiri)
A method of reacting an enzyme (ornithine cyclase) of origin (JP-A-46-27354) is known. DL
-A method for producing proline is a method in which L-proline is racemized in an acetic acid solvent using an aldehyde as a catalyst (JP-A-57-123150), and L-proline is an enzyme derived from Clostridium sticklandii (Proline). Racemase) action [Bi
ochemistry, 7 (11), 3970 (1968)] and the like are known. Further, as a method for producing D-proline from DL-proline, a method for reacting DL-proline with an L-proline-degrading bacterium to obtain the remaining D-proline (Japanese Patent Laid-Open No. Hei 4)
183399) is known. However, the manufacturing process by these methods is complicated and low in productivity, further, special equipment is required when chemical synthesis is used, and enzyme stability becomes a problem when enzymes are used. However, it is not always an industrially satisfactory method.

[0002] In addition, microorganisms having a high proline racemase activity [for example Clostridium stick Randy (Clostridium sticklandii) ATCC12662]
Was used to racemize L-proline to obtain DL-proline, and then the DL-proline produced in the aqueous medium was converted to L-proline.
-A method for producing D-proline in which D-proline remaining in the aqueous medium is collected by contacting it with a microbial cell having a proline-degrading activity, a culture solution or a treated product thereof (JP-A-4-183399). It is disclosed. However, although this method is excellent in the racemization step from L-proline to DL-proline, the Clostridium microorganism used has a low cell yield and is difficult to prepare in large quantities. Not industrially available.

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for inexpensively and efficiently producing D-proline.

The present inventors have found that the Clostridium stickland
ii ) A gene encoding the proline racemase gene of ATCC 12662 was cloned into a host microorganism, and the proline racemase activity per wet cell of the microorganism carrying the obtained recombinant DNA was Clostridium sticklandii ATCC1.
It is recognized that the L-proline is improved 23 times or more as compared with 2662, and by using the microorganism in combination with an existing microorganism having a high L-proline-degrading activity, L-proline is industrially efficiently produced by fermentation production. From this, they found that D-proline can be produced, and completed the present invention.

The present invention will be described in detail below. The present invention relates to the production of DL-proline by contacting L-proline in an aqueous medium with a bacterial cell, a culture solution or a treated product of a microorganism belonging to the genus Escherichia and having a proline racemase activity, and then producing the DL-proline in the aqueous medium. The produced DL-proline and the bacterial cells of the microorganism having an L-proline-degrading activity, a culture solution, or a treated product thereof are contacted with each other to decompose L-proline in the aqueous medium and remain from the aqueous medium. D
-A method for producing D-proline, which comprises collecting proline.

[0004] The bacterial cells of the microorganism belonging to the genus Escherichia and having a proline racemase activity, a culture solution or a treated product thereof have a pH of 4.5 to 9.5, preferably a pH of 5.5.
L-proline can be converted to DL-proline by contacting it with L-proline in an aqueous medium while maintaining 8.0. L-proline used for contacting is
Chemically pure or crude product, 10 in aqueous medium
It is used in a concentration range of ˜400 g / l, preferably 100-400 g / l. The cell concentration is 0.3 to 150 kU / l, preferably 1.5 to 30 as proline racemase activity.
kU / l is used (1 U = enzymatic activity which acts on L-proline to produce 1 μmol D-proline in 1 minute). At that time, the bacterial cells may be used as they are, but an organic solvent such as toluene or xylene may be added, or an acetone treatment may be performed. When adding an organic solvent such as toluene or xylene, the addition amount is 0.1
It may be up to 5.0% (v / v).

The pH is adjusted using an alkaline solution such as aqueous ammonia or sodium hydroxide or an acid solution such as hydrochloric acid or sulfuric acid, and the temperature during contact is 25 to 65 ° C., preferably 30 to 40 ° C. . The contact time varies depending on the amount of proline and the amount of cells used, but is usually 1 to 72 hours. DL-proline is produced in the thus obtained aqueous medium. In the aqueous medium, the produced DL-proline and the culture solution of the microorganism having L-proline-degrading activity, the cells, or the treated product of the cells were further treated to pH 6.0.
It is possible to decompose only L-proline in the aqueous medium by bringing them into contact with each other while keeping the pH at -8.0, preferably at pH 6.5 to 7.5.

The concentration of the bacterial cells used for the contact is L-
As the proline-degrading activity, 0.12 to 12 kU / l, preferably 0.6 to 1.2 kU / l is used (1 U = 1 to 1 μmol of L-proline for 1 minute to decompose the enzyme). At that time, the bacterial cells may be used as they are, but an organic solvent such as toluene or xylene may be added, or an acetone treatment may be performed. When adding an organic solvent such as toluene or xylene, the addition amount is 0.1 to 5.0.
It may be% (v / v).

The pH is adjusted with an acid solution such as phosphoric acid, hydrochloric acid or sulfuric acid at a temperature of 25 to 40 ° C. during the contact. The contact time varies depending on the amount of L-proline used in the racemization reaction and the amount of cells, but it is usually 1 to 72 hours. D-proline is present in the aqueous medium thus obtained. From the aqueous medium, microbial cells or treated cells can be removed by means such as centrifugation, and then D-proline can be collected by treatment with an ion exchange resin. The identification of D-proline is performed by measurement by high performance liquid chromatography (HPLC) and measurement of specific optical rotation.

The host microorganism transformed with the recombinant DNA containing the proline racemase gene may be any microorganism having no proline racemase activity, such as Escherichia genus, Enterobacter genus,
Examples include bacteria belonging to the genus Pseudomonas, the genus Corynebacterium, the genus Brevibacterium, and the like. These microorganisms grow well using L-proline as a sole carbon and nitrogen source, but cannot grow on D-proline because they do not have proline racemase activity. However, since the transformant strain having proline racemase activity converts D-proline to L-proline by proline racemase, it can grow well using D-proline as a single carbon source and nitrogen source. Become. Therefore,
The host microorganism transformed with the recombinant DNA is more preferably a strain lacking the restriction system and requiring L-proline.

[0009] L-proline auxotrophic strains generally cannot grow unless L-proline is present in the medium, whereas in the above microorganisms having proline racemase activity, D-drains by the action of proline racemase. -Due to its ability to convert proline to L-proline, D
-Proline shows the same growth as L-proline. Examples of L-proline auxotrophic mutants include Escherichia coli HB101 [Molecular Cloning, T. Mania.
tis et al. Cold Spring Harbor Laboratory (1982)].

The proline racemase gene source may be any microorganism belonging to the genus Clostridium and having proline racemase activity. A good example is Clostridium Stick Landy .
dium sticklandii) ATCC12662 and the like.
As the vector into which the DNA encoding the proline racemase is incorporated, any vector such as a phage vector or a plasmid vector can be used, as long as it can autonomously replicate in Escherichia spp. As a preferred example, pBR3 capable of replicating in Escherichia coli such as K12 strain
22 (manufactured by Takara Shuzo Co., Ltd.). In addition, hosts of other species such as Corynebacterium and Brevibacterium
Vector systems may also be used.

[0011] recombination with the DNA fragment and the vector encoding the proline racemase, to prepare a DNA fragment containing the proline racemase gene region by processing the chromosomal DNA of a microorganism belonging to Kurosutori di um spp an appropriate restriction enzyme, the same The recombinant DN after being incorporated into a vector treated with a restriction enzyme or a restriction enzyme which produces the same sticky end
It is carried out by transforming the host microorganism with A and isolating the transformant containing the recombinant DNA containing the target DNA fragment. This series of operations is carried out by a known in vitro recombination technique [molecular cloning (Molecular Cl
oning), T. Maniatis et al. Cold Spring
Harbor Laboratory (1982)] and the like.

A recombinant DNA containing a DNA encoding a proline racemase of a microorganism belonging to the genus Clostridium is transformed into an L-proline auxotrophic strain of a host bacterium, and D-proline is used as a single carbon source and a nitrogen source. As an auxotrophic substance, a transformant that can grow well is selected. The transformed strain is highly likely to be a strain carrying the recombinant DNA. Furthermore, by measuring the proline racemase activity of the selected strain, a strain having the target recombinant DNA can be obtained. Such transformants include Escherichia coli ( E
scherichia coli ) H-PR3. This microorganism has been deposited as FERM BP-4649 at the Institute of Biotechnology, Institute of Biotechnology, Institute of Industrial Science as of April 21, 1994.

The presence of the proline racemase gene was determined by treating the DNA fragment encoding proline racemase with various restriction enzymes to prepare small fragments, and measuring the proline racemase activity of the resulting plasmid containing each small fragment. The position can be further limited. The nucleotide sequence of the DNA fragment is determined by an automated fluorescent DNA sequencer (Applied
It can be determined by using ABI 373A manufactured by biosystems or the like.

The proline racemase activity is measured as follows. L-proline 100 g / l, toluene 2
% (V / v) in phosphate buffer (50 mM, pH
To 6.2), wet bacterial cells are added so as to be 1 to 20 g / l and reacted at 37 ° C. for 1 hour, and then the reaction solution is kept in boiling water for 10 minutes to stop the reaction. After removing bacterial cells from the reaction solution by centrifugation, the supernatant is analyzed by HPLC to measure the amount of D-proline produced.

The analytical conditions by HPLC are as follows. HPLC analysis conditions: 1) Column: TSK GEL Enantio L1 (manufactured by Tosoh Corporation) 2) Mobile phase: 2 mM CuSO ₄ aqueous solution 3) Column temperature: 40 ° C. 4) Flow rate: 1 ml / min 5) Sample amount: 20 μl 6) Detection : 254 nm UV absorption In addition, the enzyme activity which produces | generates 1 micromol D-proline in 1 minute is displayed as 1 unit (U).

As the microorganism having L-proline degrading activity, any microorganism can be used as long as it has the activity, but by using a microorganism having stronger activity, D-proline can be produced more efficiently. Such microorganisms include Candida sp.
PRD-238, and Trichosporon sp (Tri
chosporon sp.) PRD-317 (JP-A-4-18339)
9) can be given.

As a medium for culturing a microorganism having a proline racemase activity or a microorganism having an L-proline degrading activity, a medium containing a carbon source, a nitrogen source, inorganic salts and vitamins can be used. As the carbon source, various kinds of carbohydrates such as glucose, fructose, sucrose and lactose, molasses containing these and starch hydrolysates and the like are used.

As the nitrogen source, ammonium salts of various inorganic acids and organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate and ammonium phosphate, amines and other nitrogen-containing compounds, peptone, meat extract, yeast extract, Corn steep liquor, casein hydrolyzate, soybean meal hydrolyzate, various fermented cells and digested products thereof are used.

As the inorganic substance, potassium dibasic phosphate, dibasic potassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate and the like are used. The culture temperature is
20 to 42 ° C, preferably 30 to 38 ° C is used. During the culture, the pH is maintained at 5.0 to 9.0, preferably 6.0 to 7.5. The pH is adjusted using an inorganic or organic acid, an alkaline solution, calcium carbonate, ammonia or the like.

The culture time is usually 5 to 72 hours. Accumulation of proline racemase activity or L-proline degrading activity is observed in the cells of each microorganism thus obtained. Examples of the present invention will be shown below.

[0021]

【Example】

Example 1. Cloning of proline racemase gene Clostridium sticklandii ATCC1 having proline racemase
2662 was inserted into a GAM agar medium (manufactured by Nissui Pharmaceutical Co., Ltd.), and a gas pack system (Becton Dickinson and Co.,
BBL Gas Pack Anaerobic Systems, H ₂ + CO ₂ Type) was used and cultured at 37 ° C. for 2 days under anaerobic conditions.

The cultured bacterial cells were inoculated in 20 ml of GAM broth medium (manufactured by Nissui Pharmaceutical Co., Ltd.) with 1 platinum loop and statically cultured at 37 ° C. for 24 hours under anaerobic conditions using a gas pack system. Furthermore, the total amount of this culture solution is 1 liter of GA
It was transplanted to M broth medium (manufactured by Nissui Pharmaceutical Co., Ltd.) and statically cultured at 37 ° C. for 24 hours under anaerobic conditions using a gas pack system.

The culture broth was centrifuged at 4 ° C., 5,000 rpm for 10 minutes, and the cells were collected and the bacterial cells were collected in TE buffer [Tris (hydroxymethyl) aminomethane (hereinafter abbreviated as Tris) 1].
The cells were washed and collected with 0 mM, 1 mM EDTA-Na ₂ , pH 7.5], and the collected cells were subjected to the method of Saito-Miura [Biochem.Bio.
phys.Acta), 72 , 619 (1963)], high molecular chromosomal DNA was isolated.

This chromosomal DNA is treated with the restriction enzyme HindIII
Digested with. That is, a reaction solution for restriction enzyme HindIII containing ₂ μg of chromosomal DNA (Tris 50 mM, MgCl ₂
90 mM, 10 mM, 50 mM NaCl, pH 7.5)
Add 2 units of HindIII (Takara Shuzo) to 3
The reaction was carried out at 7 ° C for 60 minutes, and the reaction was stopped by heating at 65 ° C for 10 minutes.

On the other hand, pBR322 used as a cloning vector [Gene, 2 , 95 (197)
5)] was a commercially available product manufactured by Takara Shuzo. This pB
90 μl of reaction solution for HindIII containing 1 μg of R322
2 units of HindIII were added to the reaction mixture, and the mixture was reacted at 37 ° C. for 60 minutes and then heated at 65 ° C. for 10 minutes to stop the reaction.
Both reaction products were mixed, and a 10-fold concentrated buffer solution for T4 DNA ligase (Tris 660 mM, MgCl ₂ 66 mM,
Dithiothreitol 100 mM, pH 7.6) 20μ
1, 100 mM ATP (2 μl) and T4 DNA ligase (manufactured by Takara Shuzo Co., Ltd.) (350 units) were added, and ligation reaction was carried out at 12 ° C. for 16 hours. This reaction product was designated as Escherichia coli ( E
It was used for transformation of S. scherichia coli ) HB101.

Transformation was carried out by molecular cloning (Molecular Cloning, T. Maniatiset al. Cold.
Spring Harbor Laboratory (1982))
The procedure was as described. That is, L medium (containing 10 g of bactotryptone, 5 g of yeast extract, 1 g of glucose and 5 g of sodium chloride in 1 liter of water, and having a pH of 7.2).
Escherichia coli ( E
was inoculated scherichia coli) HB101, and cultured at 37 ° C. until OD _{660 nm} of 0.2. 1 in culture on ice
It was cooled for 0 minutes and then centrifuged. The cells were resuspended in 25 ml of cooled 50 mM calcium chloride-20 mM Tris, pH 8.0, placed in ice water for 15 minutes, and re-centrifuged. 2 ml of 50 mM calcium chloride-20 mM Tris,
The suspension was suspended in pH 8.0 and kept in ice for 18 hours. To 150 μl of this bacterial solution, 50 μl of the above ligase reaction mixture was added and mixed, and the mixture was placed in ice for 10 minutes and then heated at 42 ° C. for 2 minutes. Then, 2 ml of L-medium was added and the cells were cultured at 37 ° C for 2 hours. This bacterial solution was centrifuged, washed twice with physiological saline, and then ampicillin 100 μg / ml and D-proline 50 μg / ml.
M9 agar medium containing 6 ml (Na ₂ HPO ₄ 6 g, KH ₂
PO ₄ 3 g, NaCl 0.5 g, NH ₄ Cl 1 g, M
0.5 g of gSO ₄ , 2 g of glucose, 0.01 g of calcium chloride, 0.1 mg of thiamine hydrochloride were added to 1 part of water.
Medium (M9 medium) contained in 1 liter and adjusted to pH 7.2
1 liter of agar plus 20 g of medium)
Culturing was carried out at 5 ° C for 5 days. The colonies grown on the agar medium were picked up, cultured on the L medium, and the bacterial cells were collected. The proline racemase activity was examined. As a result, Clostridium sticklandii ATCC1
It was found to have a proline racemase activity almost equal to the bacterial activity of 2662. The results are shown in Table 1.

[0027]

[Table 1]

Plasmid DNA was isolated from this transformant and analyzed by restriction enzyme digestion and agarose gel electrophoresis. The size of the obtained plasmid was about 9.8 kilobases (kb), and the position of the HindIII breakpoint of pBR322 was at the position of the Clostridium sticklandi.
( Clostridium sticklandii ) It had an approximately 5.4 kb HindIII insert fragment derived from the chromosomal DNA of ATCC 12662. This plasmid was designated as pPR1 and pPR
The recombinant bacterium having 1 was designated as H-PR1. The restriction enzyme digestion map of this plasmid pPR1 is shown in FIG.

As a result of subcloning analysis of the insert fragment of pPR1, about 1.
It was found to be present in the 4 kb HindIII-EcoRI fragment. This HindIII-Eco of about 1.4 kb
The recombinant plasmid in which the RI fragment was inserted at the position of the HindIII-EcoRI cleavage point of pBR322 was designated as pPR2. pPR2 was added to Escherichia c
The recombinant strain H-PR2 introduced into oli ) HB101 is Clostridium sticki
cklandii ) ATCC12662 and H-PR1 were found to have proline racemase activity almost equivalent to that of the bacterial cells. The results are shown in Table 1. The restriction enzyme digestion map of this plasmid pPR2 is shown in FIG.

Example 2. Obtaining a strain that highly expresses the proline racemase gene During the process of subcloning pPR1, the Clostridium sticklandi ( C
lostridium sticklandii ) Hind of about 1.4 kb containing the proline racemase gene derived from ATCC 12662
III-EcoRI fragment and pPR1 that does not encode a protein
Clostridium sticklandy inserted in
A recombinant plasmid pPR3 having an approximately 1 kb HindIII-EcoRI fragment derived from ( Clostridium sticklandii ) ATCC 12662 was obtained. The process of this subcloning is shown in FIG. A restriction enzyme digestion map of this plasmid pPR3 is shown in FIG.

A recombinant strain obtained by introducing the plasmid pPR3 into Escherichia coli HB101 was designated as H-PR3. The recombinant bacterium H-PR3 is a Clostridium stick
landii ) It was found to have a proline racemase activity about 23 times as high as the bacterial activity of ATCC 12662. The results are shown in Table 2.

[0032]

[Table 2]

Example 3. Determination of the nucleotide sequence of the gene encoding proline racemase For the DNA fragment containing the proline racemase gene contained in the plasmid pPR3, the vector plasmid pUC
19, and Dele for Kilo-Sequence
About 100 bp using Tion Kit (Takara Shuzo)
Each deletion mutant strain was obtained. For this deletion mutant,
Automated fluorescent DNA sequencer (Applied biosystems)
The DNA base sequence was determined by ABI 373A), and the total DNA base sequence of the DNA fragment containing the proline racemase gene contained in the plasmid pPR3 was determined.
In this case, the sequencing kit, Taq Dye d eoxy ^TM Ter
minator Cycle Sequencing Kit (manufactured by Applied biosystems), and primers for sequencing include:
M13 Primer M4 and M13 Primer RV (Takara Shuzo) were used.

As a result, the plasmid pPR3 has
The DNA fragment containing the lorin racemase gene contains 828
ORF consisting of bp (OpenReadiongFrame) exists
It was Predicted from this base sequence and the ORF base sequence
The amino acid sequence of the protein is shown in SEQ ID NO: 1. This N end
The 20 amino acid sequence is a Clostridium stick
Randy (Clostridium sticklandii) ATCC126
62 and purified from recombinant H-PR3 cells
The sequence of the N-terminal 20 amino acids of proline racemase protein
I did it. In addition, the expected molecular weight of the protein and the
Dium stick landy (Clostridium stickland
ii) ATCC 12662 and recombinant H-PR3
SDS- of the proline racemase protein purified from
The results of molecular weight measurement by PAGE were in agreement. these
From the results, the base sequence shown in SEQ ID NO: 1
Umm Stick Landy (Clostridium sticklandi
i) A proline racemase derived from ATCC 12662
I decided.

Example 4. A recombinant strain H-PR3 carrying the racemization plasmid pPR3 of L-proline by a microorganism carrying the recombinant DNA is
300 ml L containing 100 μg / ml ampicillin
The medium was inoculated and cultured with shaking at 30 ° C. for 18 hours. After culturing, the bacterial cells were collected and added to an aqueous solution (pH 6.2) containing 100 g / l of L-proline and 1% (v / v) of toluene so that the amount of wet bacterial cells became 1 g / l (3 kU / l). In addition, add 500 ml of the reaction solution in a 1 liter Erlenmeyer flask to 37
The reaction was performed by gently shaking at 24 ° C. for 24 hours. As a result, 49.8 g / l of D-proline was produced in the reaction solution, and the amount of residual L-proline was 49.8 g / l.

Example 5. Decomposition of L-proline in DL-proline Sterilized in an autoclave, 40 ml (250 ml Erlenmeyer flask) of Candida sp. ( Candida sp.) PRD-
238 (JP-A-4-183399) was inoculated with 1 platinum loop,
Shaking culture was performed at 30 ° C. for 24 hours.

20 ml of this culture solution was sterilized in an autoclave, glucose 10 g / l, peptone 10 g /
1, meat extract 5 g / l, potassium dihydrogen phosphate 2 g /
1, magnesium sulfate 0.5g / l, ferrous sulfate 1mg
/ L, manganese sulfate 1 mg / l, and L-proline 1
Aseptically inoculate 1 liter (2 liter jar fermenter) of medium (pH 7.0) containing 0 g / l at 30 ° C.
Incubation was carried out for 24 hours with aeration and agitation (aeration amount 1 v.vm). During the culture, the medium pH was adjusted to pH 7.0 with 4N ammonia water.
It was adjusted to ± 0.1. Centrifuge 1 liter of this culture (10,000 rpm, 10 minutes) to obtain 39 g of wet cells.
Got

The reaction solution (proline racemization solution) obtained in Example 4 was diluted with distilled water so that DL-proline was 40 g / l, and the pH was adjusted to 7.0 with sodium hydroxide. Then, 1 liter of this solution was placed in a 2 liter jar fermenter and autoclaved at 120 ° C. for 20
It was sterilized for a minute and at the same time the proline racemase remaining in the solution was completely inactivated.

[0039] To this solution, Kyanjida sp (Candi
da sp.) PRD-238 live cells 30 g (1.2 kU)
And aeration stirring at 30 ° C. for 48 hours (aeration amount 1 v.v.
m.). During this time, the pH of the reaction solution was adjusted to 7.0 ± 0.1.
Was adjusted with 4N sulfuric acid. As a result of the reaction, 19.2 g / l of D-proline remained in the reaction solution, and L-proline was not detected.

Example 6. Obtaining D-proline from the reaction solution From the reaction solution of Example 5, cells were removed by centrifugation (10,0).
(00 rpm, 10 minutes), and then the supernatant liquid is ion exchange resin Diaion SK-1B (H type) (manufactured by Mitsubishi Kasei)
The column was passed through 500 ml to adsorb D-proline. After washing the resin with water, D-proline was eluted with 1N ammonia water. The eluate was concentrated under reduced pressure to obtain crude D-proline crystals 1.
4 g was obtained.

14 g of this crude D-proline crystal was dissolved in distilled water, 7 g of activated carbon was added for decolorization, and filtration was performed to obtain a filtrate. After concentrating this filtrate, it was cooled and the precipitated crystals were dried. As a result, 6 g of D-proline (optical purity 99.5 ee%
More)

[0042]

INDUSTRIAL APPLICABILITY According to the present invention, D-proline, which is important as a raw material for the synthesis of medicines and agricultural chemicals, can be industrially efficiently produced from L-proline.

[0043]

[Sequence Listing] SEQ ID NO: 1 Sequence length: 1008 Sequence type: Nucleic acid chain number: Double-stranded topology: Linear sequence type: Genomic DNA Origin organism name: Clostridium sticklandy (Clos
tridium sticklandii) Strain name: ATCC12662 Symbol representing the characteristics of the sequence: peptide Location: 1..1005 Method of determining the characteristics: S sequence ATG AAG TTT TCT AAA GGA ATT CAC GCT ATA GAT TCA CAT ACT ATG 45 Met Lys Phe Ser Lys Gly Ile His Ala Ile Asp Ser His Thr Met 1 5 10 15 GGA GAA CCT ACT AGA ATA GTT GTA GGA GGT ATT CCG CAG ATT AAT 90 Gly Glu Pro Thr Arg Ile Val Val Gly Gly Ile Pro Gln Ile Asn 20 25 30 GGA GAA ACT ATG GCT GAC AAG AAA AAA TAT CTT GAA GAT AAC CTA 135 Gly Glu Thr Met Ala Asp Lys Lys Lys Tyr Leu Glu Asp Asn Leu 35 40 45 GAC TAC GTT AGA ACT GCT TTA ATG CAT GAG CCA AGA GGA CAT AAT 180 Asp Tyr Val Arg Thr Ala Leu Met His Glu Pro Arg Gly His Asn 50 55 60 GAT ATG TTT GGT TCT ATT ATA ACT AGC TCG AAT AAT AAA GAA GCT 225 Asp Met Phe Gly Ser Ile Ile Thr Ser Ser Asn Asn Lys Glu Ala 65 70 75 GAT TTT GGA ATT ATA TTT ATG GAT GGC GGC GGA TAC TTA AAT ATG 270 Asp Phe Gly Ile Ile Phe Met Asp Gly Gly Gly Tyr Leu Asn Met 80 85 90 TGC GGC CAT GGT TCA ATT GGA GCT GCA A CT GTA GCA GTA GAA ACA 315 Cys Gly His Gly Ser Ile Gly Ala Ala Thr Val Ala Val Glu Thr 95 100 105 GGA ATG GTA GAA ATG GTA GAG CCT GTT ACT AAT ATC AAC ATG GAA 360 Gly Met Val Glu Met Val Glu Pro Val Thr Asn Ile Asn Met Glu 110 115 120 GCA CCT GCT GGA CTT ATC AAA GCT AAG GTT ATG GTA GAA AAT GAA 405 Ala Pro Ala Gly Leu Ile Lys Ala Lys Val Met Val Glu Asn Glu 125 130 135 AAA GTA AAA GAA GTA TCT ATA ACT AAC GTT CCT TCA TTT TTA TAT 450 Lys Val Lys Glu Val Ser Ile Thr Asn Val Pro Ser Phe Leu Tyr 140 145 150 ATG GAA GAT GCT AAA TTA GAA GTA CCT TCT TTA AAC AAG ACT ATT 495 Met Glu Asp Ala Lys Leu Glu Val Pro Ser Leu Asn Lys Thr Ile 155 160 165 ACA TTT GAT ATT TCT TTT GGC GGA AGC TTC TTT GCT ATT ATA CAT 540 Thr Phe Asp Ile Ser Phe Gly Gly Ser Phe Phe Ala Ile Ile His 170 175 180 GCA AAA GAG CTA GGA GTT AAA GTA GAA ACT AGC CAG GTT GAT GTA 585 Ala Lys Glu Leu Gly Val Lys Val Glu Thr Ser Gln Val Asp Val 185 190 195 CTT AAG AAA TTA GGT ATA GAA ATC AGA GAT TTA ATA AAT GAA AAA 630 Leu Lys Lys Leu Gly Il e Glu Ile Arg Asp Leu Ile Asn Glu Lys 200 205 210 ATC AAA GTT CAA CAT CCA GAG TTA GAG CAT ATC AAA ACT GTT GAT 675 Ile Lys Val Gln His Pro Glu Leu Glu His Ile Lys Thr Val Asp 215 220 225 TTA GTA GAA ATC TAC GAT GAG CCA TCT AAT CCT GAA GCT ACA TAC 720 Leu Val Glu Ile Tyr Asp Glu Pro Ser Asn Pro Glu Ala Thr Tyr 230 235 240 AAA AAC GTT GTT ATC TTT GGA CAA GGG CAA GTA GAC CGT TCT CCT 765 Lys Asn Val Val Ile Phe Gly Gln Gly Gln Val Asp Arg Ser Pro 245 250 255 TGT GGA ACT GGA ACT AGT GCT AAG CTT GCA ACA CTT TAC AAA AAA 810 Cys Gly Thr Gly Thr Ser Ala Lys Leu Ala Thr Leu Tyr Lys Lys 260 265 270 GGA CAT CTT AAG ATA GAT GAA AAA TTT GTA TAC GAA AGC ATC ACT 855 Gly His Leu Lys Ile Asp Glu Lys Phe Val Tyr Glu Ser Ile Thr 275 280 285 GGA ACA ATG TTC AAA GGA AGA GTT TTA GAA GAA ACT AAA GTT GGA 900 Gly Thr Met Phe Lys Gly Arg Val Leu Glu Glu Thr Lys Val Gly 290 295 300 GAA TTT GAT GCT ATA ATC CCA GAA ATT ACA GGG GGA GCA TAT ATA 945 Glu Phe Asp Ala Ile Ile Pro Glu Ile Thr Gly Gly Ala Tyr Ile 305 310 31 5 ACT GGA TTC AAT CAT TTC GTA ATT GAT CCA GAA GAT CCA CTT AAG 990 Thr Gly Phe Asn His Phe Val Ile Asp Pro Glu Asp Pro Leu Lys 320 325 330 TAT GGA TTT ACA GTA TAA 1008 Tyr Gly Phe Thr Val 335 SEQ ID NO: : 2 Sequence length: 335 Sequence type: Number of amino acid chains: Single strand topology: Linear sequence type: Peptide origin Organism name: Clostridium sticklandy (Clos
tridium sticklandii) Strain name: ATCC12662 Sequence features: Characteristic symbols: Location: 1..335 Sequence Met Lys Phe Ser Lys Gly Ile His Ala Ile Asp Ser His Thr Met 1 5 10 15 Gly Glu Pro Thr Arg Ile Val Val Gly Gly Ile Pro Gln Ile Asn 20 25 30 Gly Glu Thr Met Ala Asp Lys Lys Lys Tyr Leu Glu Asp Asn Leu 35 40 45 Asp Tyr Val Arg Thr Ala Leu Met His Glu Pro Arg Gly His Asn 50 55 60 Asp Met Phe Gly Ser Ile Ile Thr Ser Ser Asn Asn Lys Glu Ala 65 70 75 Asp Phe Gly Ile Ile Phe Met Asp Gly Gly Gly Tyr Leu Asn Met 80 85 90 Cys Gly His Gly Ser Ile Gly Ala Ala Thr Val Ala Val Glu Thr 95 100 105 Gly Met Val Glu Met Val Glu Pro Val Thr Asn Ile Asn Met Glu 110 115 120 Ala Pro Ala Gly Leu Ile Lys Ala Lys Val Met Val Glu Asn Glu 125 130 135 Lys Val Lys Glu Val Ser Ile Thr Asn Val Pro Ser Phe Leu Tyr 140 145 150 Met Glu Asp Ala Lys Leu Glu Val Pro Ser Leu Asn Lys Thr Ile 155 160 165 Thr Phe Asp Ile Ser Phe Gly Gly Ser Phe Phe Ala Ile Ile His 170 175 180 Ala Lys Glu Leu Gly Val Lys Val Glu Thr Ser Gln Val Asp Val 185 190 195 Leu Lys Lys Leu Gly Ile Glu Ile Arg Asp Leu Ile Asn Glu Lys 200 205 210 Ile Lys Val Gln His Pro Glu Leu Glu His Ile Lys Thr Val Asp 215 220 225 Leu Val Glu Ile Tyr Asp Glu Pro Ser Asn Pro Glu Ala Thr Tyr 230 235 240 Lys Asn Val Val Ile Phe Gly Gln Gly Gln Val Asp Arg Ser Pro 245 250 255 Cys Gly Thr Gly Thr Ser Ala Lys Leu Ala Thr Leu Tyr Lys Lys 260 265 270 Gly His Leu Lys Ile Asp Glu Lys Phe Val Tyr Glu Ser Ile Thr 275 280 285 Gly Thr Met Phe Lys Gly Arg Val Leu Glu Glu Thr Lys Val Gly 290 295 300 Glu Phe Asp Ala Ile Ile Pro Glu Ile Thr Gly Gly Ala Tyr Ile 305 310 315 Thr Gly Phe Asn His Phe Val Ile Asp Pro Glu Asp Pro Leu Lys 320 325 330 Tyr Gly Phe Thr Val 335

[0044]

[Brief description of drawings]

[0045]

FIG. 1 shows a restriction map of plasmid pPR1 containing a gene encoding a proline racemase from Clostridium sticklandy ATCC 12662.

[0046]

FIG. 2 shows a restriction map of a plasmid pPR2 containing a gene encoding a proline racemase derived from Clostridium sticklandy ATCC 12662.

[0047]

FIG. 3 shows the result of subcloning of a plasmid pPR1 containing a gene encoding a proline racemase derived from Clostridium sticklandy ATCC 12662, whereby a high expression plasmid p for proline racemase was obtained.
The process of acquiring PR3 is shown.

[0048]

FIG. 4 shows a restriction map of plasmid pPR3 containing a gene encoding a proline racemase from Clostridium sticklandy ATCC 12662.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ identification code FI (C12P 13/24 C12R 1:19) (58) Fields investigated (Int.Cl. ⁷ , DB name) C12P 13/00-13 / 24 C12N 9/00-9/99 C12N 15/00-15/90 SwissProt / PIR / GeneS eq GenBank / EMBL / DDBJ / GeneSeq BIOSIS / WPI (DIALOG) PubMed

Claims (10)

(57) [Claims] 1. An amino acid sequence represented by SEQ ID NO: 2
Containing a DNA encoding a proline racemase
Belonging to the genus Escherichia containing recombinant DNA
The microbial cell, culture solution or treated product of the microorganism to be produced is brought into contact with L-proline in an aqueous medium to produce DL-proline, and then the microbial cell having L-proline degrading activity.
The microbial cells, the culture solution or their processed products and the
A method for producing D-proline, which comprises contacting the formed DL -proline to decompose L-proline in the aqueous medium, and collecting the remaining D-proline from the aqueous medium. 2. An amino acid sequence represented by SEQ ID NO: 2
A process according to claim 1, wherein a DNA DNA encoding the proline racemase is shown in SEQ ID NO: 1. 3. An amino acid sequence represented by SEQ ID NO: 2
Belonging to the genus Escherichia containing a recombinant DNA containing a DNA encoding proline racemase
The microorganisms that make up Escherichia col
i) H-PR3 (process of claim 1, which is FERM BP-4649). 4. A racemization of L-proline to DL-proline at pH 4.5 to 9.5 and decomposition of L-proline at pH 6.0 to 8.0.
3. The method according to any one of 3 above. 5. A DNA encoding the proline racemase shown in SEQ ID NO: 1. 6. Has the amino acid sequence shown in SEQ ID NO: 2
A DNA encoding a proline racemase that 7. A recombinant DNA containing the DNA according to claim 5 or 6. 8. The recombinant DNA according to claim 7, which is a plasmid pPR3 represented by the following restriction enzyme map. [Chemical 1] 9. The recombinant DNA according to claim 7 or 8.
A microorganism belonging to the genus Escherichia, which comprises: 10. A microorganism belonging to the genus Escherichia is
Escherichia coli H-PR3
Fine of claim 9 wherein the (FERM BP-4649)
Creature .